Targeting Alanines in the Hydrophobic and Cross-Linking Domains of

Feb 22, 2018 - The analysis of the 13C-Ala line shapes, which considers sequence- and conformation-dependencies, ... The derivatization of amino acids...
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Targeting Alanines in the Hydrophobic and Cross-Linking Domains of Native Elastin with Isotopic Enrichment and Solid-State NMR Spectroscopy Jhonsen Djajamuliadi, Kosuke Ohgo, and Kristin K. Kumashiro* Department of Chemistry, University of Hawaii, 2545 McCarthy Mall, Honolulu, Hawaii 96822, United States S Supporting Information *

ABSTRACT: Details of elastin’s complex conformational and dynamic heterogeneity were acquired from one- and twodimensional solid-state nuclear magnetic resonance (ssNMR) experiments on hydrated elastin that is isotopically enriched at its alanines. Elastin’s abundant alanines are useful probes of the structural and dynamical microenvironments in its crosslinking and hydrophobic domains. High isotopic enrichment of alanines in neonatal rat smooth muscle cells (NRSMC) elastin was obtained with the inhibition of alanine transaminase, combined with an excess of [U−13C]alanine in the culture media. Due to the fast, large-amplitude motions, R-TOBSY was utilized with selective homonuclear decoupling schemes to resolve 13C-Ala peaks and confirm assignments. A data-driven approach is applied, as the interpretation of chemical shifts is based on the distribution of conformation-dependent 13C-Ala chemical shifts in proteins in multiple databases. Alanine populations in elastin’s hydrophobic domains are primarily random coil, whereas those in its cross-linking regions reside in α-helices and random coils.

1. INTRODUCTION Elastin, the primary protein component of the elastic fiber, provides extensibility and resilience to vertebrate tissues.1 One of its unique features is its composition: four amino acids, glycine (Gly, G), alanine (Ala, A), valine (Val, V), and proline (Pro, P), comprise ∼80% of the protein sample. These residues are typically arranged in repeating polypeptidyl motifs, and they form distinct hydrophobic (HP) and cross-linking (CL) domains, which are assembled in an alternating fashion.2,3 Alanine is the only amino acid type that is prominently found in both HP and CL domains of elastin.4 Thus, these abundant alanines might be exploited as useful probes of the structural and dynamical microenvironments in both of elastin’s domain types. The unique distribution of alanines is also reflected in elastin’s primary sequence. Alanines in the HP domains are generally found interspersed with other residues and are often flanked by glycine residues, such as GAG. In contrast, each Ala in the CL domains is mainly observed in tandem repeats of, e.g., KAAK or KAAAK. Nuclear magnetic resonance (NMR) spectroscopy is an effective tool for structural and dynamical studies of proteins and polypeptides in solution and in the solid state. The characterization of small soluble proteins by NMR is typically accomplished by the total assignment of all residues in the sequence. However, assignments for high-molecular-weight proteins are typically focused on select residues of interest that have been isotopically labeled. This targeted approach is best for NMR studies of a complex polymer like elastin, which can now be expressed with isotopically enriched residues. © XXXX American Chemical Society

The neonatal rat smooth muscle cell (NRSMC) culture is an effective system for the production of a native, insoluble, isotopically enriched elastin for solid-state NMR (ssNMR) investigations.5 In contrast to bacterial expression and solidphase peptide synthesis, the NRSMC line produces an insoluble elastin with native cross-links, such as desmosine and isodesmosine.6,7 Successful isotopic enrichment of the glycine residues with the NRSMC line was previously reported.3 In this initial set of studies, an equimolar substitution of the unenriched glycine with the isotopically labeled amino acid resulted in moderate enrichment with no evidence of scrambling. However, the incorporation of labeled alanines into elastin was not as straightforward, as this amino acid is produced endogenously in mammalian cells.8 In this study, we report a novel strategy for expression of NRSMC elastin with high levels of isotopically enriched alanines that exploits both exo- and endogenous pathways. One- and two-dimensional ssNMR experiments are conducted on hydrated [U-13C-Ala]elastin at the physiological temperature (37 °C). Selective detection experiments are used to identify mobile and rigid environments of Ala in the protein. The analysis of the 13C-Ala line shapes, which considers sequence- and conformation-dependencies, yields insights into the structural environments of Ala in the HP and CL domains, Received: December 8, 2017 Revised: February 22, 2018

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DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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culture media at a higher concentration than the typical formulation for NRSMC cultures, such that normal cellular metabolism is maintained, as the isotopic enrichment level in elastin is increased. The concentration of exogenous Ala that yields the highest level of isotopic enrichment was optimized. For this determination, all solutions included 1.0 mM AOA. 13C-carbonyl-enriched alanine, ([1-13C]Ala, 99%, Cambridge Isotope Laboratories, Tewksbury, MA) was supplied in the culture media at 0.5, 1.0, 2.0, and 3.0 mM. The cells were grown in a 6-well plate for 6−8 weeks, and insoluble elastin was harvested for purification. 2.3. Elastin Purification, Hydrolysis, and Amino Acid Derivatization for Liquid Chromatography−Mass Spectrometry (LC-MS) Quantification. Elastin is purified by the cyanogen bromide (CNBr) method.10 After 6−8 weeks of NRSMC culture, the harvested cell matrix was digested in 20 mL of an aqueous CNBr solution (50 mg/mL in 70% formic acid) in a 50 mL centrifuge tube for 18−24 h. Then, 20−40 mL of water were added to the mixture, which was then left under the hood for 4 h, as excess HCN gas evolves during this time. After centrifugation, the supernatant was removed, and the remaining solid was washed with water. To remove remaining contaminants, the solid was then extracted with 20 mL of 5 M guanidinium hydrochloride with 1% β-mercaptoethanol for 24 h, followed by extensive washings with water. The remaining insoluble elastin (∼120 mg) was kept in ultrapure water. The derivatization of amino acids precedes the LC-MS analysis. Purified elastin was hydrolyzed in 6 M hydrochloric acid (HCl) at 100 °C for 24 h. The elastin hydrolysate was derivatized with 1-fluoro-2,4dinitrophenyl-5-L-leucinamide (FDLA).11,12 A 250 μL aliquot of the FDLA-derivatized hydrolysate was neutralized with 150 μL of 2 M HCl and diluted with 600 μL of acetonitrile. The solution was filtered with a 20 μm disposable filter prior to LC-MS analysis. The evaluation of isotopic enrichment in alanines and detection of isotopic scrambling were performed using a tandem configuration of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) with electrospray ionization (ESI) in positive-ion mode. The separation of FDLA-derivatized elastin hydrolysate was performed on a Luna 5 μm C-18 LC column (Phenomenex, Torrance, CA). The column was regulated at room temperature, 22 °C, and an injection volume of 1 μL was used. The mobile phase consisted of a mixture of 0.1% formic acid (FA) in water (solvent A) and 0.1% FA in acetonitrile (solvent B). The elution mode consists of 30% solvent B for 35 min, followed by a linear gradient (30−50%) of solvent B, with 0.7 mL/min flow rate maintained throughout the experiment. Amino acid derivatives were detected by UV at 340 nm. Triple−quadrupole (QQQ) detection: FDLA derivatives were separated using Agilent 1200 series (Agilent Technologies, Santa Clara, CA) equipped with an autosampler, and the mass spectrometry was conducted using Agilent 6410 Triple Quad LC/MSD (Agilent Technologies, Santa Clara, CA). The ESI voltage was 4.0 kV with the gas nitrogen pressure set at 30 psi, and the capillary temperature was set to 325 °C. Data were collected in the profile mode, and a mass range of 105−1500 m/z was covered with a scan time of 500 ms. In the positive ion mode of ESI, the molecular mass of the protonated molecular ion with its most abundant, naturally occurring isotope(s) is expressed as [M + H]+, or simply [MH]+. The [MH + n]+ peak corresponds to the mass of the molecular ion with the heavier isotope(s), thus reflecting isotopic enrichment. The isotopic enrichment levels (%) of FDLA-derivatized analytes are calculated using Wolfe’s equation13

which may contribute to the overall understanding of structure−function relationships in the elastic fiber.

2. EXPERIMENTAL SECTION 2.1. Expression of Insoluble Elastin with Neonatal Rat Smooth Muscle Cell (NRSMC) Culture. The production of insoluble elastin using NRSMC culture for ssNMR spectroscopy has been reported previously.5 Briefly, 14-day timed-pregnant Sprague− Dawley rats (Charles River Laboratories, Wilmington, MA) were purchased, and the aortae from the newborn (2−3 day old) pups were extracted with aseptic technique. Following extraction, the tissue was digested with collagenase and elastase in a serum-free culture media at 37 °C for 30−45 min. The digestion period was ended by the addition of standard NRSMC media, which contains 10% fetal bovine serum (FBS). The mixture was transferred into a centrifuge tube and spun at 1000 rpm for 10 min. The pellets were suspended with (fresh) media in a centrifuge tube. This cell suspension was subsequently plated in a T-75 culture flask (Corning Inc., Corning, NY), and this primary culture was incubated in the growth media for 5−7 days to reach 70− 90% confluency. Then, a cell passage was performed, followed by secondary NRSMC culture in two T-75 flasks. After for 6−8 weeks of incubation period, the extracellular matrix (ECM) was collected using cell scrapers (Fisher Scientific, Hampton, NH) for subsequent purification of elastin. 2.2. Isotopic Enrichment Strategies. 2.2.1. Enrichment via Metabolic Precursor of Alanines. DMEM stock was purchased without glucose (Gibco, New York, NY). Uniformly 13C-enriched glucose, [U-13C, 99%]glucose (Cambridge Isotope Laboratories, Tewksbury, MA), was supplied to the cells at the typical concentration for NRSMC culture, i.e., 1000 mg/L. 2.2.2. Enzymatic Inhibition of the Biosynthesis of Endogenous (Unenriched) Ala and the Supplementation of 13C-Enriched Ala in the Culture Media. The incorporation of isotopically enriched Ala into elastin was accomplished via inhibition of endogenous Ala synthesis and the excess supply of isotopically enriched Ala in the growth media. Aminooxyacetic acid (AOA) is an inhibitor that binds to pyridoxal phosphate (PLP), i.e., the cofactor of alanine transaminase (ALT).9 This enzymatic inhibition reduces the cellular biosynthesis of Ala from pyruvate (Figure 1). Exogenous 13C-enriched Ala was supplied in the

% enrichment = 100 × (R e − R c)/1 + (R e − R c) where R, the ratio of ionic abundances (Ab) R = Ab{[MH + n]+ }/Ab{[MH]+ } is calculated for the isotopically enriched, Re, and unenriched, Rc, samples. 2.4. Solid-State NMR Spectroscopy. The approximate wet weight of a typical sample for ssNMR is 60 mg, and the water content of hydrated elastin is ∼70% (w/w). The hydrated sample was packed into a 4.0 mm rotor that was sealed with Kel-F spacers (Revolution

Figure 1. (A) Normal and (B) inhibited biosynthesis of alanine in mammalian cells. Alanine transaminase (ALT) enzyme facilitates the conversion of pyruvate to alanine. Alanine biosynthesis is inhibited, as the inhibiting agent binds with ALT’s cofactor, pyridoxal phosphate (PLP).9 B

DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Table 1. Three-Amino-Acid Sequences (R‑1-Ala-R+1) in Rat Tropoelastina R‑1-Ala-Gly group

R‑1-Ala-Z group

seq

HP

CL

B

total

seq

GAG AAG KAG VAG

25 4 1 1

5

5

35 4 3 2

AAK GAV GAL GAR GAF AAQ AAR AAS AAV AAY GAI GAP GAS KAK KAP KAQ total

total

31

1

6

1 1

7

44

HP

CL 21 2 2 1

8 3 2 2

R‑1-Ala-Ala group B 1 1

1 1 1 1 1 1 1 1 1 1 1 34

17

3

total

seq

HP

CL

21 11 6 3 2 1 1 1 1 1 1 1 1 1 1 1 54

AAA KAA PAA GAA QAA VAA LAA SAA

1

35 13 6 1 2 1 1 1

total

4 1

6

60

B

total 36 13 6 5 2 2 1 1

0

66

All 3-aa sequences for the 164 alanines in tropoelastin are listed. They are subdivided into three major groups: R‑1-Ala-Gly, R‑1-Ala-Z (Z ≠ Ala, Gly), and R‑1-Ala-Ala. For each 3-aa sequence, the number of occurrences in the hydrophobic (HP) domain, cross-linking (CL) domain, and bridging (B) regions is given. The amino acid sequence for rat tropoelastin is obtained from ELASTO-DB.4,32 a

NMR, Ft. Collins, CO), fitted with fluorosilicone micro-O-rings (Apple Rubber Products, Lancaster NY).14 The mass of the packed and assembled rotor was measured before and after each experiment to monitor the sample’s hydration level. Data were acquired on an Agilent DD2MR console (Agilent Technologies, Santa Clara, CA), equipped with a wide-bore (89 mm) superconducting magnet (Oxford Instruments, Oxford, UK), at a 1H resonance frequency of 399.964 MHz. 13C spectra were acquired using a 4 mm triple-resonance (HXY) T3 magic angle spinning (MAS) probe (Varian/Chemagnetics, Fort Collins, CO). The MAS rate was 8 kHz for all experiments. The 13C chemical shifts were referenced to the tetramethylsilane (TMS) scale, using hexamethylbenzene as an external standard (δ(13CH3) = 17.0 ppm at room temperature). All ssNMR experiments were conducted at 37 °C. The sample temperatures were calibrated using lead nitrate, Pb(NO3)2.15 For direct polarization (DP), a 4.0−4.5 μs 13C 90° pulse was used with a 6−10 s recycle delay. The 6 and 10 s delays were used for the aliphatic and carbonyl populations, respectively. These recycle delays were optimized for this sample. For cross-polarization (CP),16 a 4.5− 5.0 μs 1H 90° pulse was used with a 1 ms contact time and 5 s recycle delay. The CP transfer was set to the field strength (γHB1H/2π = γCB1C/2π) of ∼50 kHz. For {1H}-13C rINEPT,17−19 1H and 13C 90° pulses were 4.5 and 5.0 μs, respectively. Optimized delays for the first (τ−π−τ) and the second (τ′−π−τ′) spin echo sequences of rINEPT were τ = 1.2 ms and τ′ = 1.0 ms, respectively. The highest overall signal intensity is found at τ = 1.2 ms, whereas τ′ was chosen to maximize the CH intensity. A recycle delay of 1.5 s was used. Twopulse phase modulation (TPPM) 1H decoupling20 was applied during acquisition, using the field strength (γHB1H/2π) of ∼40−60 kHz. Long-observation-window band-selective homonuclear decoupling (LOW-BASHD) 21 was used for 13 CO detection with 1 J COCα decoupling. The 13C transmitter frequency was set at 175.6 ppm (on-resonance with 13CO-Ala). Selective 200 μs Gaussian 180° (π) pulses with cosine amplitude modulation were applied every 8 ms during acquisition time. The cosine modulation frequency is 12 600 Hz, which is the difference between 13CO and 13Cα-Ala resonance frequencies. Additional 4 ms delays were included before and after the first and last π pulses. The total acquisition time was 40 ms for each scan, corresponding to five cycles of π pulses. The spectral width was set to 2500 Hz, which corresponds to a 400 μs dwell time. The receiver was gated off during the first half of the dwell time (200 μs) to accommodate the π pulse; data points were consequently oversampled

in the remaining 200 μs. This oversampling results in the attenuation of FID intensities where the π pulses were applied, and the attenuation gives rise to sampling sidebands in the acquired 13CO spectrum. The sideband intensities were negligible in this study; hence, they are ignored during spectral processing. Windowed detection with selective decoupling using crafted excitation (wSEDUCE-1)22 was used to observe DP and rINEPT spectra without 1JCOCα on 13Cα-Ala and without 1JCαCβ on 13Cβ-Ala. SEDUCE-1 is a homonuclear decoupling method that uses amplitudemodulated, frequency-selective shaped pulses.23 The length of wSEDUCE-1 waveform was 638.4 μs. This waveform was placed on-resonance with 13Cα for 13Cβ-Ala detection without 1JCαCβ decoupling; it is placed on-resonance with 13CO for 13Cα-Ala detection without 1JCOCα decoupling. Phase cycling was performed using MLEV-16 supercycle.24 The dwell time (33.6 μs) was shared between the windowed acquisition and the frequency-selective irradiation. The length of the 13C irradiation was 38% of the dwell time (12.6 μs), and the highest B1 field strength among the applied pulses was γCB1C/2π = 1.1 kHz. Continuous wave (CW) 1H decoupling (γHB1H/2π ∼ 37 kHz) was used during detection. The correction to the chemical shift for off-resonance pulse effects22 for 13 Cβ with 1JCαCβ decoupling was experimentally determined to be ∼−0.1 ppm using [U-13C]Ala in H2O. The effect was not observed on the 13Cα-Ala signal, with 13CO irradiation. For the two-dimensional (2D) total bond spectroscopy (TOBSY)25 experiment, DP excitation with steady-state NOE was used to obtain the initial magnetization. The NOE was achieved using low-power 1H irradiation (γHB1H/2π ∼ 0.7 kHz) during the 3 s recycle delay. Then, a 3.9 μs 13C 90° pulse was applied, followed by TOBSY mixing with 26 Each cycle contains 30 R elements that span 6 R3014 6 symmetry. rotor periods. Two successive R elements are phase-shifted by 84° and −84°. 13CO−13Cα and 13Cα−13Cβ correlations were obtained from two separate experiments, which employ the respective mixing times (τmix) of 8.9 and 6.0 ms or 12 and 8 R cycles. No 1H decoupling was applied during the mixing period. 13C carrier frequencies during the mixing time were placed at 34.4 and 113 ppm for the respective 13 Cα−13Cβ and 13CO−13Cα correlations. Spectral widths in the indirect dimensions were 5700 and 5200 Hz for 13Cα−13Cβ and 13 CO−13Cα correlations, respectively, with the center of the window at 48.0 ppm. During t1, WURST-2 decoupling27 was applied to simultaneously remove both 1JCOCα and 1JCαCβ, which affect the 13CαAla line shape. The WURST-2 decoupling waveforms were calculated C

DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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Figure 2. 13C DPMAS spectra of hydrated elastin samples from NRSMC cultures (A) in standard (unenriched) media, (B) with [U-13C]glucose, and (C) with excess [U-13C]Ala (180 mg/L) and 1.0 mM AOA to inhibit ALT. In (A−C), dotted lines indicate signals from 13C-Pro and 13C-Gly, and dashed lines indicate 13C-Ala intensities. Spectrum A reflects the natural-abundance 13C signals from all amino acids in NRMSC elastin. The number of acquisition scans for (A), (B), and (C) was 1024, 256, and 256, respectively. using Pbox software (Agilent). The pulse width and bandwidth for WURST-2 on each carbon were set as 4.0 ms and 1000 Hz, respectively. The highest B1 field strength of the pulse was γCB1C/2π ∼ 0.8 kHz. The TPG-5 supercycle (0°, 150°, 60°, 150°, and 0°)28 was applied to the selective pulses, as described in the literature.29 The correction for off-resonance pulse effects22 of ∼−0.1 ppm was applied to the F1 dimension. Homonuclear decoupling during acquisitions of 13 Cα−13Cβ and 13CO−13Cα correlations was achieved using wSEDUCE-1 and LOW-BASHD, respectively. 128 scans were acquired per t1 point. The total number of t1 complex points for 13 Cα−13Cβ and 13CO−13Cα correlations was 115 and 105 points, respectively, corresponding to the maximum t1 length of 20.0 ms. Total measurement times for these experiments were 15.5 and 16.5 h, respectively. 2.5. Semiempirical Prediction of the 13CO Line Shape for Alanines in Tropoelastin with the Fully Random Coil State. The semiempirical approach17 was employed to predict 13C chemical shifts for fully (100%) random coil alanines in NRSMC elastin. Briefly, these calculations were based on the 13C chemical shifts of random coil Ala, obtained from the solution NMR study of the model peptide AcGGXGG-NH2 in 8 M urea, where X represents one of 20 amino acids.30 The sequence dependence, or neighboring residue effect, of Ala (A) in the three-amino-acid motif, R−1-Ala-R+1, was computed by

domain, cross-linking (CL) domain, and bridging (B) regions is given. In the case of the latter, the 3-aa sequence runs across HP and CL domains. In addition, the chemical shift of Ala in each 3-aa sequence was then plotted with a Gaussian function (fwhm = 0.17 ppm), with relative intensity that reflects the number of occurrences of that sequence in tropoelastin. This 13CO-Ala simulated line shape, with −2.65 ppm offset applied, was compared to the skyline projection of observed 13CO−13Cα cross-peaks. The major contribution to the offset (−2.65 ppm) is the difference in chemical shift references between DSS and TMS, i.e., −2.11 ppm. The remainder, ∼−0.54 ppm, is explained by the differences in experimental conditions (solvents and temperatures) between the calculated and the observed spectrum. As described, the semiempirical prediction was based on the NMR study of the model peptide Ac-GGAGG-NH2 in 8 M urea at 20 °C, whereas the spectrum for hydrated elastin (in water) was acquired at 37 °C. This minor, systematic, offset allows for best comparison between the calculated and observed line shape.

3. RESULTS AND DISCUSSION 3.1. Combination of ALT Inhibition and Excess 13CEnriched Ala in the Culture Media Yields the Highest 13C Enrichment Level for Alanines in NRSMC Elastin. Endoand exogenous pathways were explored for the labeling of elastin’s alanines. In the case in which the focus was the endogenous pathway, 13C-enriched glucose was included in the prepared culture media, replacing the unenriched sugar in the culture media. Another strategy combined enzymatic inhibition with an excess of 13C-enriched alanines in the growth media, utilizing exogenous and endogenous pathways. LC-MS was used to quantify the 13C enrichment levels of alanines in NRSMC elastin that were grown under different conditions. U-13C-glucose supplementation resulted in ∼50%

δAla(corrected) = δrc(Ala) + Δδ(R −1) + Δδ(R+1) where δrc(Ala) is the base value for random coil Ala in urea and Δδ(R−1) and Δδ(R+1) are correction factors that account for preceding (R−1) and following (R+1) residues.31 13CO-Ala chemical shifts were calculated systematically for each of the R−1-Ala-R+1 sequences found in rat tropoelastin (Table 1). Table 1 gives a full listing of the three-amino-acid sequences (R−1-Ala-R+1) in rat tropoelastin. They are subdivided into three major groups: R−1-AlaGly, R−1-Ala-Z (Z ≠ Ala, Gly), and R−1-Ala-Ala. For each 3-aa sequence, the number of occurrences in the hydrophobic (HP) D

DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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Figure 3. 1D 13C MAS ssNMR spectra of hydrated [U-13C-Ala]elastin at 37 °C. DP, rINEPT, and CP line shapes are shown in black, green, and red, respectively. The sum of (CP + rINEPT) intensities are shown in magenta. CP and rINEPT intensities were adjusted, such that the difference between the traces for the DP spectrum and the sum of (CP + rINEPT) is minimized. LOW-BASHD decoupling during acquisition of 13CO was used to remove 1JCOCα. Windowed SEDUCE-1 during DP and rINEPT acquisitions decouples the homonuclear interactions between 13CO and 13 Cα (1JCOCα) on 13Cα-Ala and those between 13Cα and 13Cβ (1JCαCβ) on 13Cβ-Ala regions. The correction for off-resonance pulse effects of ∼+0.1 ppm was applied to the 13Cβ-Ala spectra. Asterisks in 13CO and 13Cα line shapes indicate natural-abundance Gly signals.

and the corresponding breakdown of 3-aa sequences that characterize either the hydrophobic or cross-linking domains provide the foundation for the interpretation of multiple resonances. 3.2.1. 1D MAS NMR Experiments Identify Mobile and Rigid Environments of Alanines in NRSMC Elastin. Solid-state NMR (ssNMR) spectroscopy is suitable for the characterization of alanines in hydrated, insoluble elastin.5,10,33−36 Direct polarization (DP) with refocused insensitive nuclei enhanced by polarization transfer (rINEPT) and cross-polarization (CP) experiments under magic angle spinning (MAS) provide isotropic 13C (or 15N) chemical shifts, which are useful for the assignment of secondary structures. In addition, rINEPT and CP experiments selectively detect the mobile and rigid regions in elastin, respectively.17 For these reasons, DP, rINEPT, and CP spectra of hydrated [U-13C-Ala] elastin were acquired with homonuclear decoupling scheme at 37 °C to provide a complete picture of the protein at physiological temperatures. The DP spectra reflect all carbon populations, regardless of conformation and dynamics (Figure 3, black traces). All 13C sites in hydrated elastin are not observed with the typical 13C CPMAS NMR experiment. Thus, DP is employed to obtain the spectrum that contains all 13C nuclei in this protein. Asymmetric line shapes are observed for all 13C sites, indicating multiple Ala populations in NRSMC elastin. The narrow line widths are similar to those observed in previous NMR studies of hydrated elastin, reflecting fast, large-amplitude, and nearly isotropic motion across the sample.5,35,36 The 13CO signal has a narrow line shape (∼125 Hz) that has highest intensity at 175.9 ppm, with a downfield shoulder centered at ∼177 ppm. The 13 Cα signal appears homogeneous, as only one broad line shape (∼145 Hz) is observed with the tallest point at 50.5 ppm. The highest 13Cβ intensity is identified at 17.4 ppm with an upfield shoulder centered at ∼16.5 ppm. 13CO, 13Cα, and 13Cβ resonances at 175.9, 50.5, and 17.4 ppm, respectively, are consistent with the statistical average of Ala’s chemical shifts for random coil.37,38 Resonances at ∼177 ppm (13CO) and ∼16.5 ppm (13Cβ) are consistent with alanines in α-helices.39,40 The 13C-Ala line shapes produced by the sum of CP and rINEPT intensities (Figure 3, magenta traces) are consistent with those acquired by DP. The 13C-Ala rINEPT spectra reflect the mobile regions in NRSMC elastin (Figure 3, green traces). Only 13Cα- and 13Cβ-Ala peaks were observed by rINEPT; no 13 CO-Ala signal was detected due to the absence of directly

isotopic enrichment at the alanines; i.e., 50% of the alanines in elastin carried the 13C label. The strategy which combined inhibitor with enriched Ala in the media yielded 13C-Ala enrichment levels that varied with (Ala) concentration. In the presence of 1.0 mM AOA (inhibitor) in the culture media, the enrichment level in samples grown in 0.5, 1.0, 2.0, and 3.0 mM [1-13C]Ala were ∼40%, ∼55%, ∼ 80%, and ∼70%, respectively (Supporting Information, Figure S1). Within the noted uncertainties, the enrichment levels with 2.0 and 3.0 mM [1-13C]Ala are roughly equivalent. As such, the incorporation of [1-13C]Ala was optimal at 2.0 mM. The high isotopic enrichment of alanines via exo- and endogenous pathways is reflected by the 13C intensities in the ssNMR measurements (Figures 2A−C). Elastin that was expressed in the standard, or unenriched, media shows lowest relative intensities (Figure 2A). The sample produced with [U-13C]glucose shows prominent 13C peaks for Ala, Pro, and Gly residues. The glucose is a precursor to these and other amino acids, but these peak intensities are highest, due to the abundance of these small hydrophobic amino acids in elastin (Figure 2B). Although the enrichment and corresponding NMR peak intensities are high, this nonspecific labeling strategy is not optimal for the targeted characterization of alanines in elastin. Among several complications, the most notable is the significant peak overlap. For instance, the 13CαAla peak at ∼50 ppm overlaps with the 13Cδ-Pro intensity at ∼49 ppm. NRSMC elastin that was expressed in 1.0 mM AOA and 2.0 mM [U-13C]Ala showed the highest signal intensities for the targeted amino acid (Figure 2C). The 13CO-, 13Cα-, and 13 Cβ-Ala peaks are identified at ∼176, ∼50, and ∼17 ppm, respectively. Furthermore, the natural-abundance 13C intensities of other amino acids are negligible. In addition, there is no evidence for isotopic labeling of the other (non-Ala) amino acids. In most cases, the synthesis of the amino acids is unaffected by the presence of the inhibitor and/or excess Ala. LC-MS assays directed toward the ones that may be affected (Glx, Asx, Arg, Pro) indicate negligible levels of isotopic enrichment, particularly as the low levels of Glx and Asx are considered. 3.2. Solid-State NMR Studies Describe the Conformational Heterogeneity of Alanines in NRSMC Elastin in a Domain-Specific Fashion. NMR experiments in one and two dimensions were used to observe multiple populations of alanines in elastin. 13C chemical shifts are analyzed using elastin’s unique composition and comparisons to structural database information; i.e., the primary structure of tropoelastin E

DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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Figure 4. (A) 13C DP spectra of hydrated [U-13C-Ala]elastin at 37 °C and (B) their deconvolution results; (C) distributions of 13C chemical shifts of alanines in RefDB,41 categorized as HELIX, SHEET, and COIL. In (A), the homonuclear-decoupled 13CO-Ala line shape was acquired using DP with LOW-BASHD.21 Homonuclear decoupling for 13Cα- and 13Cβ-Ala line shapes is achieved using wSEDUCE-1.22 MAS is applied during the entire experiment, and CW or TPPM heteronuclear decoupling was applied during the acquisition for wSEDUCE-1 and LOW-BASHD experiments, respectively.The asterisk marks the natural-abundance 13CO-Gly signal. In (B), deconvolution subroutine is performed with Lorentzian−Gaussian (L/G) components, and the sum of peaks identified by deconvolution is shown in magenta. Vertical dashed lines in (B) (and (C) show center-ofmass for each peak as identified by deconvolution. Histograms in (C) illustrate the distributions of 13C chemical shifts of Ala in the corresponding structures. 13C-Ala resonances are sorted and categorized into intervals (or bins) spanning 0.2 ppm. The height of each bar reflects the relative number of observations of a given bin of 13C-Ala resonances, normalized over the total number of occurrences. The sum of the bar heights is 1 (or 100%) in each histogram.

rereferenced and corrected using SHIFTX,50 a program that calculates NMR resonance frequencies from the X-ray or NMR atomic coordinates. These 13C chemical shifts for alanines in these 2100+ structures are then categorized as HELIX, SHEET, or COIL (Figure 4C). The volume, area, and dihedral angle reporter (VADAR)51 server assigns the approximate secondary structure (HELIX, SHEET, COIL) using a weighted combination of three popular structural recognition approaches that utilize geometrical constraints,52 dihedral angles,53 and hydrogen-bonding patterns54 as criteria. VADAR’s recognition pattern for HELIX (or SHEET) follows a defined set of torsion angles and hydrogen-bonding networks; structures that meet the criteria for neither HELIX nor SHEET are classified as COIL. For instance, all types of β-turns are included in COIL. In general, 13CO- and 13Cα-Ala chemical shifts in a HELIX are found in the downfield region of the range of observed peaks, whereas those corresponding to SHEET are upfield. The 13CβAla chemical shifts show the opposite trend. The RefDB assignment of 13C-Ala populations in elastin (Table 2) reflects the likelihood that a population corresponding to a given resonance resides in the HELIX, SHEET, or COIL distribution. For example, alanines with the carbonyl resonance of 175.9 ppm are found in the chemical shift distribution for COIL with 5.6% probability (or 0.056), corresponding to the bin height in the histogram. These alanines also have 0.031 and 0.022 probability for HELIX and SHEET, respectively. Hence, COIL is the most likely designation for the 13CO-Ala signal at 175.9 ppm in elastin’s DP spectrum. The certainty of this assignment is expressed as COIL (0.056) > HELIX (0.031) > SHEET (0.022), based on the bin intensities at the corresponding chemical shift in each structural distribution.

bonded protons. The tallest intensities in 13Cα- and 13Cβ-Ala line shapes were observed at 50.5 and 17.3 ppm, respectively, which are consistent with the random coil assignment.37,38 This mobile environment is presumably described by fast largeamplitude fluctuations, which have motional time scales that are compatible with observation by rINEPT, τc < 10−9 s (recalculated for ∼9.4 T).41,42 Such fluctuations may be related to structural interconversions described for statistical coils.43−47 In contrast, the 13C-Ala CP spectrum reflects the relatively rigid regions in elastin (Figure 3, red). The absolute CP intensities are significantly lower than the rINEPT and DP spectra due to reduced dipolar couplings in the hydrated sample. The 13CO line shape is broad (∼400 Hz), and its tallest point is observed at 176.0 ppm. The centers of masses in 13Cα- and 13Cβ-Ala signals in the CP spectra are identified at 50.5 and ∼17 ppm, respectively. 3.2.2. 13C Chemical Shifts Are Compared against Database Information To Identify Multiple Populations of Alanines with Predominantly Random Coil Character with Significant Contributions from Helical and Sheetlike Structures. A deconvolution subroutine was performed on the DPMAS spectrum to identify distinct populations of alanines in NRSMC elastin (Figure 4A,B). Each of the three 13 C sites in Ala was deconvolved, using Lorentzian−Gaussian (L/G) functions. The deconvolution subroutine yields a data set of parameters that describe the peaks that fit the line shape, including chemical shift, line width, and area or intensity. These values were compared to 13C chemical shift distributions of alanines in proteins that are archived in RefDB48 (Table 2). RefDB is a database of protein chemical shifts derived from the biological magnetic resonance data bank (BMRB).49 It contains 1H, 13C, and 15N chemical shifts of 2162 proteins (as of September 2016). The chemical shifts in RefDB have been F

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Multiple components are necessary to deconvolve the 13C DP spectrum of elastin. The 13CO signal requires a minimum of six components to reproduce the experimental result (Figure 4B). The major contribution 1 (43%) at 176.5 ppm most likely corresponds to alanines in HELIX (0.052); VADAR assigns the HELIX structure to peaks with this shift most frequently, with lesser designations to COIL (0.031) or SHEET (0.009) (Table 2). Components 2, 3, and 4 contribute to the total Ala population in elastin by 20% (175.9 ppm), 6% (175.4 ppm), and 11% (175.0 ppm), respectively. These resonances are most consistent with the 13C chemical shift distributions of COIL. Additionally, these three components reflect the neighboring residue effects in the 13CO-Ala chemical shifts, which are discussed in greater detail in the next section. Another component, 5 (12%) at 174.4 ppm, is assigned as SHEET (0.048). Component 6 originates from the natural abundance 13 CO-Gly signal, with no structural designation. Two components adequately reproduce the 13Cα-Ala line shape. The minor component 7 (9% of total line shape) centered at 52.2 ppm is associated with both HELIX and COIL populations, but a relatively higher probability is found for the former (0.033) than latter (0.029). Such comparable bin intensities may also indicate that a mixture of structures is often found for Ala at the corresponding chemical shift. In contrast, the major contribution 8 (91%) best matches 13C-Ala distributions of COIL (0.077) in RefDB. The HELIX component (7) in this 13Cα-Ala line shape, 9%, is significantly less than that in the 13CO-Ala (43%) described previously. The discrepancy may be due to 1JCαCβ line shape broadening (wSEDUCE-1 decoupling on 13Cα only removes the 1JCOCα), which affects the fitting process during deconvolution subroutine. The 13Cβ-Ala line shape was fitted using three components, no. 9 centered at 17.6 ppm, no. 10 at 17.3 ppm, and no. 11 at 16.7 ppm. Lines 9 and 10 are most consistent with the COIL

Table 2. Results of Deconvolution Subroutine with RefDB Structural Assignmenta site 13

13

13

CO

Cα

Cβ

no.

δ (ppm)

width (Hz)

area (%)

1

176.5

293

43

2

175.9

46

20

3

175.4

45

6

4

175.0

53

11

5

174.4

160

12

6* 7

172.1 52.2

148 171

7 9

8

50.4

156

91

9

17.6

63

27

10

17.3

39

22

11

16.7

151

51

structural assignment with RefDB HELIX (0.052) > COIL (0.031) > SHEET (0.009) COIL (0.056) > HELIX (0.031) > SHEET (0.022) COIL (0.078) > SHEET (0.033) > HELIX (0.015) COIL (0.054) > SHEET (0.035) > HELIX (0.007) SHEET (0.048) > COIL (0.038) > HELIX (0.005) HELIX (0.033) > COIL (0.029) > SHEET (0.003) COIL (0.077) > SHEET (0.042) > HELIX (0.007) COIL (0.060) > SHEET (0.034) > HELIX (0.020) COIL (0.075) > HELIX (0.029) > SHEET (0.028) COIL (0.075) > HELIX (0.060) > SHEET (0.021)

a13

CO-, 13Cα-, and 13Cβ-Ala chemical shifts correspond to the center of masses of each peak identified by the deconvolution of DP spectra with LOW-BASHD21 and wSEDUCE-122 decoupling. Structures are classified based on VADAR’s assignments of 13C chemical shifts of alanines in 2162 proteins in RefDB.41 HELIX and SHEET represent the α-helix and β-sheet structures, respectively. COIL indicates other motifs, which are neither α-helix nor β-sheet. Numbers in parentheses, e.g., HELIX (0.052 or 5.2%), reflect the probability that alanines at a given resonance are observed in the distribution of the designated structure; these numbers correspond to the bar intensities in the histograms. The structure of each component identified in the deconvolution is assigned based on these probabilities. The asterisk marks the natural-abundance 13CO-Gly signal.

Figure 5. Two-dimensional R-TOBSY spectra of hydrated [U-13C-Ala]elastin at 37 °C. (A) 13CO−13Cα and (B) 13Cα−13Cβ correlations. Top: DPMAS spectra reproduced from Figure 1 (black) and skyline projections (blue) are shown for comparisons. Bottom: 2D cross-peaks reflect alanine populations in NRSMC elastin. Guidelines (dashed lines) indicate Ala populations reflected in the contour peaks of 13CO−13Cα and 13Cα−13Cβ correlations, which are consistent with the center of masses of components in the 13CO-Ala line shape deconvolution (1, 2, 3, and 4 in Table 2). WURST-2 decoupling during t1 period yield 13Cα line shapes without 1JCOCα and 1JCαCβ in F1 dimensions of both (A) and (B). LOW-BASHD and wSEDUCE-1 yield line shapes without 1JCOCα and 1JCαCβ in the respective F2 dimensions of (A) and (B). The correction for off-resonance pulse effects of ∼−0.1 ppm was applied to the F1 dimension (in A and B). The correction for off-resonance pulse effects of ∼+0.1 ppm was applied to the F2 dimension (B). G

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shifts to query the Protein structure And Chemical Shift NMR spectroscopY (PACSY) database (of >3000 proteins)57 and outputs the possible amino acid types and the associated secondary structures. This program was originally designed for resonance assignments and structural analyses of disordered or large proteins, which typically feature broad line widths. The query input is obtained from pairwise chemical shift correlations, e.g., 13Cα−13Cβ, 13CO−13Cα, and 13CO−15N, which are acquired from multidimensional ssNMR spectra under MAS. In essence, the structural assignment approach using PLUQin parallels that of RefDB/VADAR described previously. The PLUQin program combines common and statistically rare secondary structures in the PACSY database into three categories: helix includes α-, 310-, and π-helices, sheet includes β-sheet and isolated β-bridge, and coil includes random coil and turns. The program yields structural assignments as percentages (%), reflecting the relative number of alanines found in each defined structure (helix, coil, or sheet) in the 3000+ proteins in the database.57 Elastin structures were predicted using observed 13CO-, 13Cα-, and 13 Cβ-Ala chemical shifts that were rereferenced to DSS (+2.1 ppm), required by PLUQin. PLUQin predicts α-helix and random coil for the major populations of alanines in NRSMC elastin (Table 3). In the

designation, as the bin intensities (0.060 and 0.075, respectively) are the highest and line widths are small (∼50 Hz). Line 11 also has the highest intensity for COIL (0.075), but its probability for HELIX (0.060) is comparable. Notably, the widths for downfield 13CO and 13Cα populations are greater than those that are likely found in the coil regions. Therefore, the 13Cβ at 16.7 ppm is tentatively assigned to the alanines in helical regions, as it is most upfield and it has a broader line width. 3.2.3. 2D R-TOBSY Confirms the Assignments of the Three Populations of Mobile Alanines, and Comparisons to Database Information Identify Predominant Random Coil Character in These Regions. The deconvolution subroutine and the comparison of 13C-Ala chemical shifts with RefDB histograms suggested the existence of multiple Ala populations in common motifs, such as α-helix and β-sheet. However, unambiguous structural assignments could not be achieved from the 1D analysis, as the number of peaks identified in the deconvolution were inconsistent among the 13C-Ala sites. For example, components 1, 7, and 11 appear to originate from the α-helical population, based on the RefDB assignment (shown in Table 2), even though the corresponding areas obtained by deconvolution subroutine are inconsistent. Hence, two-dimensional (2D) ssNMR spectroscopy was used to resolve overlapping 13C-Ala signals. The 2D spectra yield chemical shift correlations for all 13C-Ala sites, which may be used to identify secondary structures. High-resolution 13CO−13Cα and 13 Cα−13Cβ correlation spectra were acquired using R-TOBSY with WURST-2 decoupling during t1 and LOW-BASHD and/ or wSEDUCE-1 during acquisition (t2). These decoupling procedures aimed to remove the J-couplings, 1JCOCα and 1JCαCβ, which reduce spectral resolution in the direct and indirect dimensions (Supporting Information, Figure S2). The homonuclear correlation spectra ( 13 CO− 13 Cα, 13 Cα−13Cβ) include multiple alanine signals. Overlapping peaks are observed in both 13CO−13Cα (Figure 5A) and 13 Cα−13Cβ correlations (Figure 5B). The highest intensities span broad ranges in the regions expected for 13CO (∼174.5− 176.5 ppm), 13Cα (∼49.5−51.6 ppm), and 13Cβ (∼16.5−18.0 ppm). The three most intense features, 2, 3, and 4, correspond to the random coil population of alanines. Their respective centers of masses are identified at (13CO, 13Cα) = (175.8 ppm, 50.6 ppm), (175.3, 50.3), and (175.0, 50.1), which are associated with the respective (13Cα, 13Cβ) correlations at (50.6, 17.2), (50.3, 17.4), and (50.1, 17.6). A minor signal, 1, is also observed with (13CO, 13Cα) and (13Cα, 13Cβ) correlations at (176.6, 51.5) and (51.5, 16.6), respectively, which corresponds to the α-helical region. The attenuated crosspeak intensity of the α-helical population is due to the short spin−lattice relaxation time-constant in the rotating frame (T1ρ), which reduces the transfer efficiency during TOBSY mixing (i.e., during each R30 sequence). In general, the 2D homonuclear correlation experiments yield results that are consistent with the components identified with deconvolution, as described in the previous section. Identification of the 13C chemical shifts via 2D ssNMR spectroscopy facilitates the conformational assignment of alanines in NRSMC elastin. Secondary structures for each of the resolved populations were predicted using an automated resonance assignment program, PLUQin,55 an enhanced version of PACSYlite Unified Query (PLUQ).56 PLUQ is a Python-based program that takes the input of protein chemical

Table 3. Predicted Conformations of Alanines in NRSMC Elastin Using PLUQin55 a Ala, δ (ppm) alanine population 1 2 3 4

13

CO

176.6 175.8 175.3 175.0

PLUQin prediction (%)

13

13

Cβ

helix

coil

sheet

51.5 50.6 50.3 50.1

16.6 17.2 17.4 17.6

37.4 2.0 1.0 0.7

61.4 91.6 91.3 88.5

1.2 6.4 7.7 10.8

Cα

a

Predictions were based on the resolved chemical shifts of alanines from the 2D 13C−13C correlation spectra of Figure 5. PLUQin sorts common and statistically rare secondary structures found in the PACSY database into three groups: helix includes α-, 310-, and πhelices; sheet includes β-sheet and isolated β-bridge; and coil includes random coil and turns.55 Each structural classification (helix, coil, and sheet) is associated with a range of chemical shifts in the database. For each set of observed correlations (of [U-13C-Ala] elastin), Ala’s propensity for a given structural classification is expressed as a percentage (%), reflecting the relative number of occurrences for a given conformation.

PACSY database, alanines that have 13CO, 13Cα, and 13Cβ chemical shifts corresponding to population 1 in elastin, i.e., 176.6, 51.5, and 16.6 ppm, respectively, are frequently as observed as helix (37.4%) and coil (61.4%). These shifts are found in the overlapping regions of the (chemical shift) distributions for helix and coil.57 Population 1 has lower relative mobility or slower time scales of motion, as evident in its reduced intensity in Figure 5. Defined secondary structures, such as an α-helix or β-sheet, would likely experience slower motions than, e.g., those in an unstructured or “random coil” environment. In this regard, we note that there is no evidence for β-sheets or similar structures, which would be observed in the upfield regions for 13CO, for example. When considering the questions of heterogeneity and multiple conformations, we note that the helix designation itself accounts for α-helices as well as the less common π- and 310-helices; i.e., this broad grouping also entails structural heterogeneity. H

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Macromolecules Chemical shifts for Ala populations 2 and 3 are coil (∼92%), with residual amounts of helix (1−2%) and sheet (6−8%). Interestingly, PLUQin also gives the identical percentages for 13 C-Ala chemical shifts of denatured elastin, i.e., coil (91− 92%), helix (1−2%), and sheet (6−7%) (Supporting Information, Table S1), indicating that populations 2 and 3 in hydrated elastin are most likely random coil. Population 4 is also best described as nonhelical, as the corresponding chemical shifts (13CO, 13Cα, 13Cβ = 175.0, 51.5, 17.6 ppm) are almost never observed in the helix (0.7%), but primarily in coil (88.5%) and secondarily in sheet (10.8%). The PLUQin results, based on 2D 13C−13C correlations, are consistent with the RefDB assignments of Table 2, which were obtained for the components of the 1D 13C-Ala line shapes. 3.2.4. Characteristic 3-aa Sequences of the Hydrophobic and Cross-Linking Domains Are Correlated with the Four Major Populations of Alanines and Their Most Likely Conformations, Based on Chemical Shift Data. The origin of three distinct intensities describing the random coil population of alanines in the previously described 2D 13 CO−13Cα cross-peaks was further examined by a semiempirical method.17 This approach aimed to predict the 13COAla chemical shifts of a fully (100%) random coil state and to compare them with the observed resonances. The base 13CO resonance value for the random coil Ala was obtained from the study based on a model peptide Ac-GGXGG-NH2 in 8 M urea, where X represents one of 20 amino acids.30 Then, correction factors31 that incorporate the effect of preceding (R−1) and following (R+1) residues on the carbon resonances of Ala in the three-amino-acid-sequences, R−1-Ala-R+1, were calculated systematically for each of the sequences found in rat tropoelastin (Table 1). Using this approach, the three random coil Ala populations (Figure 6, black) are identified as R−1-Ala-Gly, R−1-Ala-Ala, and R−1-Ala-Z (Z ≠ Ala, Gly). The greatest 13CO intensity at 175.0 ppm corresponds to the R−1-Ala-Ala sequences, primarily found in the cross-linking domains (Table 1). The peak at 175.9 ppm arises from the R−1-Ala-Gly sequences, which are abundant in the hydrophobic domains. The smallest contour peak observed at 175.3 ppm, corresponds to R−1-Ala-Z sequences (Z ≠ Ala, Gly), found in both hydrophobic and cross-linking domains. The preceding residues, R−1, exert very little of the neighboring residue effect on the 13CO-Ala chemical shifts.17 Features in the skyline projection of calculated line shape (black) are consistent with the skyline projection of observed spectrum (blue), as the centers of masses of R−1-Ala-Gly, R−1-Ala-Z, and R−1-Ala-Ala signals roughly match the experimental peaks at 175.8, 175.3, and 175.0 ppm, respectively. The relative peak intensities in the observed spectrum are not identical with the calculated ones. Components 3 and 4 in the experimental cross-peaks are significantly lower in intensity than population 2, but the three calculated signals (R−1-AlaGly, R−1-Ala-Z, and R−1-Ala-Ala) have comparable heights. This result indicates that alanines in NRSMC elastin do not adopt a fully random coil state, which is reflected by the calculated cross-peaks. A minor random coil population is identified for alanines in the cross-linking domains (3, 4), as indicated by the calculated R−1-Ala-Ala and R-Ala-Z signals (Table 1). The remainder of Ala signals originating from the cross-linking regions are presumably found in the more rigid αhelical population (1) at 176.6 ppm, which would not be clearly detected by the 2D R-TOBSY experiment.

Figure 6. Comparison of experimental and calculated spectra for alanines in NRSMC elastin. Top: skyline projections for experimental cross-peaks (blue) and calculated line shape (black) are shown for comparison. Components 2, 3, and 4 indicate alanines in the mobile region of NRSMC elastin, previously identified by deconvolution subroutine (Figure 4). Bottom: experimental 13CO−13Cα cross-peaks (blue) are reproduced from Figure 2 using a higher contour threshold. Semiempirical prediction (black) indicates three alanine populations in the fully random coil state, with R−1-Ala-Gly, R−1-Ala-Ala, and R−1Ala-Z (Z≠ Ala, Gly) sequences (see Experimental Section). Vertical dashed lines indicate the centers of masses of calculated signals, which are consistent with the observed intensities.

The conformation and sequence dependencies of 13C chemical shifts play substantially into the assignments of the ssNMR signals for alanines in elastin (Table 4). Alanines with R−1-Ala-Ala sequences in the cross-linking domains are found in both α-helix (population corresponding to peak 1) and random coil populations (peak 4). In contrast, mobile alanines in R−1-Ala-Gly motifs, which are largely found in the hydrophobic domains, are found as random coil (2). The population characterized by R−1-Ala-Z motif (Z ≠ Ala, Gly) is distributed across both hydrophobic and cross-linking regions, and it also corresponds to random coil (3) and possibly in helical domains (1).

4. CONCLUSION Solid-state NMR studies of alanines in elastin offer insights into the protein’s molecular organization, as this amino acid is found throughout its sequences and is abundant in both hydrophobic and cross-linking domain types. The study necessitates the use of elastin samples with high levels of isotopic enrichment at the alanines. The presence of endogenous Ala in NRSMC limits the incorporation of isotopically enriched Ala supplied in the growth media into elastin. For this reason, alternative strategies that incorporate Ala into elastin via endo- and exogenous pathways were investigated. The optimal isotopic enrichment scheme involves the inhibition of ALT to minimize the concentration of endogenous (unenriched) Ala, while concurrently providing excess isotopically enriched Ala to the I

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Macromolecules Table 4. Alanine Populations in NRSMC Elastina Ala population

relative mobility

predominant structure

R−1-Ala-R+1 group

representative sequence

domain type

1 2 3 4

low high high high

α-helix random coil random coil random coil

R−1-AA, R−1-A-Z R−1-AG R−1-A-Z R−1-AA

AAA, KAA, AAK GAG, AAG, VAG GAV, GAL, AAK AAA, KAA, PAA

CL HP HP + CL CL

a

1, 2, 3, and 4 correspond to Ala populations of Table 2 and were confirmed by 2D NMR experiments (Figure 5 and Table 3). Sequence information was obtained from ELASTO-DB4,32 (Table 1). The PLUQin program was used to predict the probability of finding the site in a helix, sheet, or coil by comparing 13C chemical shift to solved structures in the PACSY database. HP and CL represent hydrophobic and cross-linking domains, respectively.

commonly noted.67 Most recently, the juxtaposition of the hydrophobic effects and conformational entropy as driving forces for restoration (in elasticity) and aggregation were explored in a computational study of aggregates of the elastin mimetics of the hydrophobic domains,68 complementing a related NMR study of aggregates of an elastin mimetic.69 Results of the current study are consistent with these findings, namely, that the hydrated and native protein is characterized by the predominance of unstructured, fast-moving segments with regions of defined secondary structure, even if transient in nature. Future studies will further explore the nature of this dynamic structural heterogeneity, as it relates to biological elasticity.

growth media. In the presence of 1.0 mM AOA and 180 mg/L [U-13C]Ala in the culture media, the biosynthesis of alanine was inhibited, and a high level of isotopic enrichment (∼80%) in elastin was achieved with minimal isotopic scrambling. Solid-state NMR measurements of hydrated [U-13C-Ala]elastin at 37 °C provide a description of the microenvironments of Ala in the protein at physiological temperatures. Assignments of secondary structures were made by isotropic 13C-Ala chemical shifts obtained via one- and two-dimensional ssNMR experiments. The asymmetric line shapes in 1D 13C spectra suggest the presence of multiple Ala populations in NRSMC elastin, which is also supported by features in the cross-peaks of 2D 13C−13C correlation spectra. Alanines in the cross-linking domains are characterized by α-helix and random coil, whereas those in hydrophobic domains are primarily found as random coil. The random coil population includes Ala from the cross-linking domains, as confirmed by the sequence dependence or neighboring residue effect for 13C chemical shifts. This effect was confirmed by a semiempirical approach that gave rise to three 13C-Ala signals corresponding to the R−1-AlaGly, R−1-Ala-Z (Z ≠ Ala, Gly), and R−1-Ala-Ala sequences found in rat tropoelastin. The prediction was found in good agreement with the observed 2D 13C−13C correlation spectrum. The result also indicates that only a small portion of Ala in cross-linking domains is found in random coil, whereas the remainder are helical. The assessment of Ala’s mobility in elastin was conducted using selective detection experiments, i.e., CP and rINEPT. The α-helical population of Ala was observed primarily by CP, which is sensitive to slower motions with τc > 10−4 s (recalculated for ∼9.4 T).41,42 Such motions presumably correspond to the dynamics of Ala in the α-helical segments, which include helix-breathing and -bending.58 In contrast, the random coil population of Ala was selectively detected by rINEPT, which is sensitive to faster motions, i.e., τc < 10−9 s (recalculated for ∼9.4 T).41,42 Such motions may be attributed to large-amplitude fluctuations found in both native elastin36 and its mimetics,59−61 as also found in the flexible loop regions of globular proteins.62,63 In conclusion, these ssNMR studies provide new details of the structure distribution of alanines in native, hydrated NRSMC elastin at 37 °C. Alanines in the hydrophobic domains are predominantly random coil with a smaller amount of nonhelical secondary structures, whereas the cross-linking domains are characterized by a mixture of helices and random coil. This complex structural profile is consistent with the earliest models for elastin’s structure−function, namely, the liquid drop64 and random network;65 the models for elastin have evolved to include more detail from experiments on peptides modeled after single domains of elastin,60,66 giving rise to the conformational ensembles or structure distributions

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02617. Figures S1 and S2; Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone 1-808-956-5733; Fax 1-808-956-5908; e-mail [email protected]. ORCID

Kristin K. Kumashiro: 0000-0002-5208-1119 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grants MCB-1022526 and CHE1532310.

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REFERENCES

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DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.7b02617 Macromolecules XXXX, XXX, XXX−XXX